We examined the effect of polymorphonuclear cells on the release of tumor necrosis factor (TNFalpha) in endotoxin-treated macrophages. Human peripheral blood neutrophils were co-cultured with mouse peritoneal macrophages stimulated with lipopolysaccharide (LPS). In a dose-dependent manner, FMLP (n-formyl-methionyl-leucyl-phenylalanine) augmented the release of TNFalpha by LPS-stimulated macrophages in the presence, but not in the absence, of neutrophils. The stimulating effect of neutrophils on macrophages was reversed by catalase, suggesting that the release of hydrogen peroxide from neutrophils was responsible for augmenting macrophage TNFalpha. Moreover, the direct addition of hydrogen peroxide to macrophages resulted in an increased secretion of TNFalpha. In addition, insertion of a porous membrane between the neutrophils and macrophages cancelled the effect, indicating that adherence of neutrophils may be necessary for augmentation of TNFalpha release. In summary, the data suggest that hydrogen peroxide released from stimulated neutrophils may act as an activator of macrophage function by increasing their release of TNFalpha.